Molecular Biology 2016
Assignment 1
Dilutions and Concentrations
Only submit the answers to one digit after the decimal, you do not need to show your
calculations
(1 point/question)
1. You add water to 125 mL of 1.6 M LiCl to obtain 1.0 L of solution, what is the new
concentration of LiCl?
2. In the previous problem, the solution contains how many parts of solute and of solvent?
3. You start with 2.5L of a KNO3 stock solution and wish to prepare 10.0 L of 1.5 M KNO3.
What molarity would the potassium nitrate stock solution need to be if you were to use it all?
4. How many milliliters of 5.0 M copper sulfate solution must be added to 228 mL of water to
achieve a 0.25 M copper sulfate solution?
5. 40.0 mL of 2.0 M Fe(NO3)3 is mixed with 2 mL of 5 M Fe(NO3) and 48 mL of water. What
is the final molar concentration of Fe(NO3)?
6. You add 3.5 L of an HCl solution of unknown concentration to 2.0 L of 0.5 M HCl and
obtain a solution with a final concentration of 1.5 M. What was the unknown concentration
of the initial HCl solution?
7. You have a solution representing 126g/L of NaF (MW: 42g/mole). 180 mL of this solution is
added to water to obtain a final volume of 1080 mL. What is the molarity of the resulting
solution?
8. What is the molar concentration of chloride ions in a solution prepared by mixing 100.0 mL
of 2.0 M KCl with 50.0 mL of a 1.50 M CaCl2 solution?
9. A solution is prepared by dissolving 54 g of AgNO3 in 156 mL of water. What is the percent
concentration (m/m) of AgNO3 of this solution?
10. The A260nm of a DNA solution is 0.12. How much of this DNA solution and a 5.5X loading
dye should you add to 15µL of water to obtain a sample which contains 30ng of DNA in
0.5X loading dye? (A260nm of 1.0 = 50 µg/mL DNA)
1.
2.
3.
4.
5.
0.2M
1 part solute & 7 parts of solvant
6M
12 mL
1M
6. 2.1M
7. 0.5M
8. 2.3M
9. 25.7
10. 5µL DNA and 2µL dye
Molecular Biology 2016
Restriction Enzymes & Restriction Mapping
(3 points/question)
11. The nomenclature of restriction enzymes can provide useful information about the source of
the enzyme. For example, EcoRI indicates that this enzyme was the first enzyme isolated from
an E.coli strain “R”. From which bacteria was PvuII isolated from?
Proteus vulgaris
12. Define the following terms: Isoschizomer, neoschizomer, and isocaudomer.
Isoschizomers: restriction enzymes specific to the same recognition sequence and cut in the same
location.
Neoschizomer: An enzyme that recognizes the same sequence but cuts it at a different position.
Isocaudomer: An enzyme that recognizes a different sequence, but produces the same ends.
13. Amongst the enzymes listed below, which if any, generate compatible ends to each other? (Ex.
A and B)
Enzyme
Recognition Sequence
Cut
EcoRI
5'GAATTC
5'---G AATTC---3'
BamHI
5'GGATCC
5'---G GATCC---3'
HindIII
5'AAGCTT
5'---A AGCTT---3'
TaqI
5'TCGA
5'---T CGA---3'
NotI
5'GCGGCCGC
5'---GC GGCCGC---3'
Sau3AI
5'GATC
5'--- GATC---3'
PvuII
5'CAGCTG
5'---CAG CTG---3'
KpnI
5'GGTACC
5'---GGTAC C---3'
PstI
5'CTGCAG
5'---CTGCA G---3'
SacI
5'GAGCTC
5'---GAGCT C---3'
SalI
5'GTCGAC
5'---G TCGAC---3'
ScaI
5'AGTACT
5'---AGT ACT---3'
SpeI
5'ACTAGT
5'---A CTAGT---3'
SphI
5'GCATGC
5'---GCATG C---3'
StuI
5'AGGCCT
5'---AGG CCT---3'
XbaI
5'TCTAGA
5'---T CTAGA---3'
BamHI – Sau3AI
PvuII-ScaI-StuI
SpeI-XbaI
Molecular Biology 2016
14. The linear 12 Kbp DNA fragment shown below has cleavage sites for BamHI and EcoRI. The
numbers indicate the distance in kilobases. Complete the table to indicate the fragment sizes
which would be observed on an agarose gel following each of the indicated digests. Note, if
different fragments of the same size are generated, the size should only be indicated once. (For
example do not indicate 2Kbp and 2Kbp)
B
E
B
E
1
4
6
10
Enzyme digest
Fragment sizes
BamHI
1, 5, and 6Kbp
EcoRI
2, 4, and 6Kbp
BamHI + EcoRI
1, 2, 3, and 4Kbp
15. What fragment sizes could be generated from a BamHI partial digest? Only indicate the sizes
of intermediate fragments which would not be obtained following a complete digest.
(Fragments which contain one or more BamHI site which remains undigested)
12, 6 and 11 Kbp
16. Consider the results of the BamHI digest you indicated in the above table. Draw all possible
maps, (including the one illusrated above) which could correspond to the results indicated for
the BamHI digest alone.
(1,5,6), (6,1,5), (5,6,1), (6,5,1), (5,1,6), (1,6,5)
17. It was determined that the enzyme XhoI cuts at 2.0Kbp on the map shown above. Indicate
which BamHI fragment would be cut by XhoI and what sizes would be generated in each of
the maps provided for question 16. (Ex. 4Kbp BamHI → 3 + 1 Kbp).
5→1+4, 6→2+4, 5→2+3, 6→1+5
18. A complete digest with EcoRI + BamHI of 12µg of the above fragment was performed.
Indicate the amount in µg of each of the fragments which would be obtained.
1 µg of 1Kbp
4 µg of 2Kbp
3 µg of 3Kbp
4 µg of 4Kbp
Molecular Biology 2016
19. The circular 10Kbp DNA molecule shown below has cleavage sites for BamHI and EcoRI.
Complete the table to indicate the fragment sizes which would be observed on an agarose gel
following each of the indicated digests. Note, if different fragments of the same size are
generated, the size should only be indicated once. (For example do not indicate 2Kbp and
2Kbp)
10/0 Kbp
EcoRI
BamHI 3 Kbp
6 Kbp BamHI
Enzyme digest
Fragment sizes
BamHI
3 and 7Kbp
EcoRI
10Kbp
BamHI + EcoRI
3 and 4Kbp
20. What fragment sizes could be generated from a BamHI partial digest? Only indicate the sizes
of intermediate fragments which would not be obtained following a complete digest.
(Fragments which contain one or more BamHI site which remains undigested)
10Kbp
21. Consider that the EcoRI site is invariable. Is this the only possible map according to the results
presented in the above table? If not draw another possible map.
EcoRI
7 Kbp BamHI
BamHI 4 Kbp
Molecular Biology 2016
22. It was determined that the enzyme XhoI cuts at 9Kbp on the map shown above. Indicate which
BamHI fragment would be cut by XhoI and what sizes would be generated in each of the maps
from questions 19 and 21. (Ex. 4Kbp BamHI → 3 + 1 Kbp).
7→3+4
9→2 + 7
23. An 8.9 kb circular plasmid is digested with three restriction enzymes, EcoRI, BamHI and
HindIII, individually and in combination, and the resulting fragment sizes are determined by
means of electrophoresis. The results are as follows:
EcoRI
BamHI
HindIII
EcoRI + BamHI
EcoRI + HindIII
BamHI + HindIII
BamHI + EcoRI + HindIII
8.9 kb
6 kb. 2.9 kb
8.9 kb
6 kb, 2.4 kb, 0.5 kb
7.4 kb, 1.5 kb
5 kb, 2.9 kb, 1 kb
5 kb, 2.4 kb, 1 kb, 0.5 kb
Draw a possible restriction map based on these results. Set the EcoRI site as the origin.
Any rotation and mirror image of this map are valid.
8.9/0 Kbp
EcoRI
8.4 Kbp BamHI
BamHI 2.4 Kbp
7.4 Kbp HindIII
Molecular Biology 2016
24. Obtain the picture of the agarose gel electrophoresis of digests of the plasmid pBR322. (The
file can be found on the course’s web site under the rubric “Sequences>pBR322”. Based on
the results obtained, answer the following questions:
a. How many times did PvuII cut within the plasmid?
once
b. How many times did HincII cut within the plasmid?
twice
c. How many times did HincII cut within the PvuII fragment?
twice
d. What are the distances between the PvuII and the HincII sites?
Approx. 1415 & 1840
Lab exercise
(4 points/question)
Dilutions exercise with micropipettors (Pg. 13)
1. Indicate the absorbance readings obtained for each of the following solutions which you
prepared in the first lab exercise. If you are submitting the assignment as a group of 2,
indicate the average absorbance for both samples.
a.
b.
c.
d.
e.
A 1.5mM solution of compound “A”.
A 0.36% (m/v) solution of compound “B”.
A 6% (v/v) solution of solution I.
A solution containing 0.5mg of compound “A” and 0.1% (v/v) of compound “B”.
A solution representing the following ratio: solution I: solution II : water : 2:1:2
Should be within 25% of the following values to obtain points:
a. 0.285
b. 0.094
c. 0.15
d. 0.075
e. 1.1
Determining DNA concentration: (Pg. 14)
2. Submit a table of the DNA concentration determinations experiment performed on page 14 of
the lab manual. Your table should include the following information: Standard DNA
concentrations (µg/mL), corresponding A260 readings, and A260 readings of each of the
unknown diluted solutions of DNA you prepared.
Check general directives. Verify that absorbance readings are consistent with the
dilution.
3. Submit a graph representing the A260 readings Vs standard DNA concentrations. Include a
line of best fit, the R coefficient, and the formula of the line.
Check general directives. R2 coefficient should be greater than 0.95
4. Determine from your graph, what DNA concentration in µg/mL corresponds to an A260 of 1.0.
Accept anything in agreement with their graph
5. According to the constant determined in the previous question, what was the DNA
concentration of the undiluted unknown DNA solution provided?
Accept anything in agreement with their ans. for previous question
Molecular Biology 2016
Restriction digests & agarose gel electrophoresis (Pg. 15-17)
6. Submit a figure and an appropriate figure legend of the agarose gel described on page 17 of
the lab manual. Check general directives.
7. Submit a standard curve of the molecular weight ladder (Migration distance Vs. Size in Kbp)
Check general directives.
8. Submit a table of the restriction digests of the recombinant plasmid which includes the
following information: Enzyme used, number of cuts, fragment sizes observed.
Check general directives.
9. In a caption accompanying the table submitted, indicate the total size of the plasmid, the size
of the vector, the size of the insert, and the restriction site (s) in which the insert was introduced
in the vector.
Check general directives.
10. Provide a figure which represents a possible restriction map of the insert within the multiple
cloning site of pUC9. Your map should be linear and only include the insert within the multiple
cloning site. (See directives on this course’s web site)
E
B
P
H
B
E
0.6Kb 1.2Kb
H
2.0Kb
Molecular Biology 2016
Bioinformatics 1
11. Submit a table with the following information with regards to each of the unknown genes from
the first bioinformatics exercise. (6 points)








The accession number (#2)
Coverage
Ident.
E value
The definition (#1)
The organism from which this sequence was obtained (#3)
The product of the gene (#4)
The protein id. This is the protein’s accession number (#5)
Accession number
Coverage
Ident
E value
Definition
Organism
Gene
product
Protein id
NM_011882
100%
100%
0.0
Mus musculus
ribonuclease L
Mus musculus
Rnasel
NP_036012.1
XM_001122124
100%
100%
0.0
Apis mellifera
blue
XP_001122124.2
AY154834
30%
100%
0.0
AAN86870.1
100%
100%
0.0
Drosophila
mojavensis
Homo sapiens
Adh-1
NM_005109
OXSR1
NP_005100.1
X01108
25%
100%
0.0
Apis mellifera
bluestreak (blue)
alcohol
dehydrogenase 1
oxidative stress
responsive 1
cytochrome
oxidase subunit II
(CO II)
Triticum
aestivum
cytochrome
oxidase
subunit II
CAA25581.1
12. Submit a printout of a FASTA sequence of one of the unknown genes. Include the name of
the gene as a heading to the printout. (2 points)
Give points if provided
Molecular Biology 2016
Assignment #2
Restriction digests and mapping (4 points/question)
The table below presents the results of different digests of a plasmid.
HindIII
3.82, 0.18
BamHI
4.0, 2.35, 1.65
EcoRI
3.0, 1.0
HindIII + BamHI
3.55, 2.35, 1.2, 0.27, 0.18
HindIII + EcoRI
1.87, 1.0, 0.95, 0.18
BamHI + EcoRI
1.6, 1.4, 1.0, 0.75, 0.25
1. What is the total plasmid size? 4Kbp
2. Which of the digests is (are) inconsistent with the other results? Give a possible explanation
for the inconsistency indicating which fragment size (s) is (are) in disagreement. Partial; the
size adds up to more than the total size of 4Kbp. 4Kbp band is a partial.
3. Draw a circular map which is in agreement with the results presented.
4. Given that 1µg of DNA was used for the EcoRI digest, what are the approximate quantities in
µg of each of the fragments?
3.0Kbp: 0.75µg and 1.0Kbp: 0.25µg
5. The restriction enzyme ApoI cleaves the sequence R/AATTY (R= A or G and Y = C or T).
How many different palindromes does ApoI recognize? 2
6. A DNA fragment generated with the restriction enzyme XbaI (T/CTAGA) was inserted into
the unique NheI (G/CTAGC) site of a vector. Indicate the new 6 base sequence generated
following ligation of the XbaI site to the NheI site. Accept either GCTAGA or TCTAGC
Molecular Biology 2016
7. Could the new recombinant plasmid described in the previous question be digested with XbaI,
NheI, or both enzymes to release the insert? Could not be release with any.
You performed restriction digests and agarose gel electrophoresis of a plasmid using 3 different
restriction enzymes. The gel is shown below. Unfortunately, you forgot to label your tubes. The
only things you remember is that your standards are in Lane 5 and your uncut control is in Lane 1.
Also, you loaded the same amount of total DNA in all the sample wells (1-4).
1
2
3
4
5
*
16Kb
8 Kb
2.5 Kb
2.0 Kb 2
1.0 Kb
0.5 Kb
8. What is the approximate size of the plasmid?

20 kb

16 kb

6.5 kb

5.0 kb
9. How many restriction sites are there in the plasmid for the enzyme used in Lane 2?
2
10. How many restriction sites are there in the plasmid for the enzyme used in Lane 4?
3
11. What is the most probable size of the band labelled with a star in lane 4?
14kbp
Molecular Biology 2016
PCR
Consider the following information to answer questions 12-15: (4 points/question)
You wish to amplify a 1 Kbp single copy sequence from 1 µg of a single stranded genome which
is 109 Kbp using a pair of primers “A” and “B”.
12. What is the minimum number of cycles required to obtain a double stranded amplification
product delimited by primers “A” and “B”? 3
13. What mass of the double stranded PCR product (in µg) delimited by primers “A” and “B”
would you have after the number of cycles indicated in the previous question? 2 x 10-9 µg
14. What mass of the double stranded PCR product (in µg) delimited by primers “A” and “B”
would you have after a total of 30? Approx. 0.27 µg
15. How many additional PCR cycles would be required to attain the same yield of product which
was obtained with 1 µg of genomic DNA if you had started with 1 ng of genomic DNA?
Approx. 10
Consider the following information to answer questions 16-19: (4 points/question)
Below is the partial sequence of a 2Kbp region of DNA you wish to amplify and clone in the vector
pST18. Below the sequence is indicated the restriction information for the sequence you wish to
amplify as determined from a bioinformatics analysis as well as the MCS of pST18.
Number
of cuts
Position
BamHI (G/GATCC)
2
26 & 1615
BclI (T/GATCA)
0
Restriction enz.
EcoRI (G/AATTC)
0
pST18
SfaA1 (GCGAT/CGC)
1
3.0 Kbp
XbaI (T/CTAGA)
2
540
500 & 1500
16. The following sequence represents that of one of the primers designed to amplify and clone
the desired sequence: 5'-GAATTCAGGGTCGGCTAT-3'. The underlined sequence represents
an EcoRI restriction site. Indicate the sequence of the first 20 bases of the PCR product which
would be synthesized from this primer (including the primer sequence)
5'-GAATTCAGGGTCGGCTATCC
Molecular Biology 2016
17. Design a second 15 bases primer, which in combination with the one described in the previous
question would allow the amplification of the desired region. Include in your primer sequence
a restriction site which would allow the directional cloning of the sequence of interest. (Note,
the restriction site must be additional to the 15 bases. Underline the sequence of the restriction
site and indicate its identity.
BclI
5’-T/GATCA GACTGAATGCCACAC
18. Your primers allowed you to amplify a 2 Kbp sequence. You digest the amplicon and 100ng
of the vector with the appropriate enzymes in order to clone the sequence of interest. How
much of the digested amplicon should be added to the ligation mix in order to have an insert
to vector ration of 3:1? 200ng
19. Following the ligation, you isolate plasmid DNA from a potential recombinant. What digest
could be used to verify the presence and the orientation of the desired insert. Indicate the
enzyme (s) and the fragment size (s) expected if this is the desired recombinant.
XbaI: 3.5, 1, and 0.5Kb
Molecular Biology 2016
Lab exercise
Project I: Verifying the restriction map of a DNA insert (4 points/question)
1. Submit a figure representing the agarose gel electrophoresis of your single digests. Make sure
to include an appropriate legend. Follow the directives for figures on the web page of this
course. Make sure to include all the required information in the legend for the understanding
and interpretation of the figure.
Check general directives. Should be similar to this depending on the orientation they had:
1.
2.
3.
4.
5.
6.
7.
8.
Mol wt. marker
Uncut
BamH1
HindIII
PstI
PvuII
ScaI
pUC Bam
2. Submit a figure representing the agarose gel electrophoresis of your double digests. Make sure
to include an appropriate legend. Follow the directives for figures on the web page of this
course. Make sure to include all the required information in the legend for the understanding
and interpretation of the figure. Check general directives.
Molecular Biology 2016
3. Submit a table presenting the analysis of the restriction digests. Your table should
include: Enzyme (s) used, Total number of cuts, Number of cuts in the vector, Number
of cuts in the insert, and Fragments sizes generated. Check general directives.
4. Submit a figure of the restriction map of the insert. Your map must be linear, include
the multiple cloning site, indicate the insertion site, the size of the insert, the positions
in the multiple cloning site or the insert of all the enzymes tested. Your figure must be
to scale. Follow the directives for generating such a figure under the heading
Graphs/Figures on this course's web site. Check general directives.
Bioinformatics 2 (1 point/question)
Restriction mapping
5. Present theoretical maps of all unknown genes. Indicate below each map the name of
the gene and list the enzymes which do not cut. Give points if done and required
info is given.
6. Compare the theoretical maps generated above to the experimental map of the
unknown insert you analyzed in Project I. The unknown insert corresponds most
closely to which gene? CoxII
7. Submit a printout of the FASTA sequence of the gene corresponding to the unknown
insert. Give points if done
8. Indicate how many times each of the following enzymes cut within the unknown insert
identified in question 6: AccI (2), BglII (0), MboI (18), NcoI (1), and NotI (0).
9. Amongst the enzymes indicated in the previous question, which one cuts the most
often within the DNA insert? Give a reason which would explain why this enzyme
cuts more often than the others. MboI, recognition sequence is only 4 bases
therefore more probable than others which are 6 - 8 bases.
Molecular Biology 2016
Bioinformatics 3 (1 point/question)
10. What is the predicted size of the GFP product amplified with the primers used in lab exercise
2? 487bp
11. Submit a figure showing the positions and directions of each of the following primers on the
pUC19 sequence.
A. TGCGGTGTGAAATACCCT
B. GCCATTCAGGCTGCGCAA
C. GGGTTATTGTCTCATGAG
D. GAGACAATAACCCTGATA
Indicate in a legend to your figure all primer pairs, if any, would give an amplification product
of at least 200bp.
A.187
C.2523 D.2535
B.245
2686
B and D give a product of at least 200bp
12. Present the complement, inverse, inverse complement and the complement of the inverse of
the following sequence:
5’- GAATGCGGCTTAGACTGGTACGATGGAAC-3’
Complement: 3’CTTACGCCGAATCTGACCATGCTACCTTG
Inverse: 5’CAAGGTAGCATGGTCAGATTCGGCGTAAG
Inverse complement: 5’GTTCCATCGTACCAGTCTAAGCCGCATTC
Complement of the inverse: 3’ GTTCCATCGTACCAGTCTAAGCCGCATTC
Molecular Biology 2016
Assignment #3
(3 points/question)
1. You decide to clone your favorite gene
(yfg) into a 6000bp target plasmid that
contains an inducible promoter. To
achieve high expression of yfg, you must
clone yfg in the same orientation as the
promoter in the target vector
(arrowheads must point in the same
direction). You have already mapped the
plasmid for three sticky-end restriction
enzymes as shown (assume a negligible distance between the sites in the target vector’s
multiple cloning site within lacZ). You also note that yfg has a HindIII site that cuts yfg into
a 200bp fragment and an 800bp fragment in your TetR vector when double digested with
HindIII and EcoRI. What enzyme(s) would you use to cut the target vector to clone yfg into
it? EcoRI
2. Using your strategy, will yfg insert into the target vector in the same orientation as the promoter
always, sometimes, or never? Sometimes
3. Which single restriction enzyme can be used to conclusively confirm that yfg is cloned in the
correct orientation in the target vector? Indicate what results you expect to obtain if yfg is in
the correct and in the incorrect orientation.HindIII
Shown below is a restriction map for the beta-globin gene from mouse. You digest mouse
genomic DNA with EcoRI (GAATTC) and resolve the digestion products on an agarose gel.
The DNA is then denatured and probed with different labelled fragments for a Southern
analysis.
1.0Kb
1.0Kb
4.0Kb
0.8Kb
Beta globin gene
Probe 2
Probe 1
4. What fragment size (s) would you expect to see if you perform a Southern blot on the agarose
gel using the labelled DNA probe 1 indicated on the figure? 0.8 and 4 Kbp
5. What fragment size (s) would you expect to see if you perform a Southern blot on the agarose
gel using the labelled DNA probe 2 indicated on the figure? Give the most accurate estimate
if a precise size cannot be determined. 1 and approx. 4 Kbp
Molecular Biology 2016
6. You cloned a genomic DNA fragment generated from an EcoRI digest into a unique EcoRI
site of a vector. You identify a recombinant vector that you believe has the DNA of interest.
Suppose you find that the gene of interest is in the vector, but now you want to generate a
restriction map of the recombinant plasmid. You take three individual samples of the plasmid
and digest them with EcoRI, HindIII, and with both EcoRI and HindIII. You then run the
digested DNA on an agarose gel to see the fragments. The gel is then subjected to a Southern
hybridization using the genomic EcoRI fragment as a probe. Assuming the genomic fragment
is smaller than the vector, which fragments on the gel would hybridize to the probe?
EcoRI
4.1
2.7
1.5
1.1
0.4
HindIII
EcoRI + HindIII
5.3
4.1
2.7
3.0
2.3
1.5
1.1
0.4
7. Draw the restriction map of this recombinant plasmid.
Molecular Biology 2016
Refer to the following description to answer questions 8-11
You designed an experiment to clone a 0.8Kb fragment digested with EcoRI and HindIII into the
unique EcoRI and HindIII restriction sites of a vector. A double digest of the vector generates two
fragments of 7.2 Kb and 0.016Kb. In order to minimize reassembly of the vector, the 0.016Kb
fragment was removed from the double digest preparation of the vector. Transformations of the
following samples were then performed:
a.
b.
c.
d.
5pg of undigested vector
30ng of EcoRI digested vector which was treated with DNA ligase
30ng of EcoRI + HindIII digested vector which was treated with DNA ligase
30ng of EcoRI + HindIII digested vector mixed with 10ng of the digested DNA
fragment and then treated with DNA ligase
Below is indicated the number of colonies obtained for the different samples. Unfortunately,
the plates representing transformations b-d were not labelled.
Undigested vector
50
I
II
III
45
350
0
8. What was the ratio between insert : vector in reaction “d”? 1 : 3
9. What was the transformation efficiency with the undigested vector? (transformants/µg)
1 x 107
10. Which plate has the highest probability of containing colonies representing plasmid
recombinants? I
11. Which plate illustrates intramolecular ligation events? II
Molecular Biology 2016
12. Fred is married to Helen, who was previously married to George, now deceased. George and
Helen conceived one child together and adopted one child. Fred and Helen have also
conceived one child. All members of Helen’s current family have had DNA fingerprinting
done at a single VNTR locus. Unfortunately, the sheet that identified each child has been
misplaced. Identify which fingerprint in each lane (in lanes 5, 6, and 7) correspond to each
child.
Fred and Helen’s conceived child
Child __2__
George and Helen’s conceived child Child __3__
George and Helen’s adopted child
Child __1__
13. Considering all the individuals tested on the gel in the previous question, what is the minimum
number of different alleles which were observed of the VNTR? 4
14. If Fred was the father of the adopted child, what would be a possible profile for the VNTR of
the mother? She would have band 1 only or band 1 plus any other band
15. The following diagram shows a Southern blot of restriction
digested genomic DNA from an elf Mother (M) and elf
Father (F) and four potential elf children (C1 to C4) probed
with a VNTR DNA sequence. The restriction enzyme used
was Not I. Another elf (F2) is claiming to be the father of
child C4. Assume the mother, M, is the real mother of
these four children. Which children are biologically related
to the elf Father (F)? C1, 2 and 4.
16. Which children if any, could be elf F2's children? C3
17. Consider the data in the lane labelled “markers”.
According to this lane only, what is the minimum number
of loci which are being examined? 13
18. If the number of loci indicated in the previous question represents the total number of loci
being examined, the profile of C4 must include a minimum of how many heterozygous loci?
3 loci
Molecular Biology 2016
Lab Exercises
(4 points/question)
Genomic DNA fingerprinting
1. Submit a figure of the agarose gel representing the analysis of the ApoC2 gene. Include an
appropriate legend indicating the sizes observed.
2. Submit a figure of the agarose gel representing the analysis of the ApoB gene. Include an
appropriate legend indicating the sizes observed.
3. Provide an analysis of the genomic fingerprints obtained overall in the class. Your analysis
should include the following information:



Number of different alleles observed
Frequency of occurrence of each allele
Number of individuals homozygous and heterozygous for each of the alleles
Project II: Amplification and mutagenesis of GFP
4. Submit a figure representing your PCR of the GFP gene. Include an appropriate legend
indicating the expected size as determined by bioinformatics and the experimental size,
determined from your gel.
Transformation and screening of GFP recombinants
5. Indicate the number of transformants obtained following the transformation of the ligation
mixtures with and without the GFP amplicon. Submit your data as colony counts/mL
6. Submit a figure of the digestions performed on the amplicons obtained by colony PCR.
7. Submit a table which presents the following information for each of the 4 clones you screened:

Digestion with SalI: yes or no
Molecular Biology 2016
Bioinformatics 4 (1.5 points/question) (Give points if done and required info is given)
8. Submit a table indicating the following information:







Human alpha 1 nucleotide accession number
Human alpha 2 nucleotide accession number
Human alpha 1 protein accession number
Human alpha 2 protein accession number
Baboon nucleotide accession number
Baboon protein accession number
The percentage identity between each pair of nucleotide homologues and each pair of
protein homologues.
9. Between the two most closely related proteins, indicate the percentage conserved, semi
conserved or not conserved amino acid substitutions with respect to the total number of
substitutions?
10. Submit a table indicating the following information:



The source organism of each of the protein homologues examined in part II of the bioinfo
exercise
Their accession numbers
The percentage identity between each pair of protein homologues.
11. With respect to the first protein homologue, what type of homologue is each of the other protein
homologues?
Molecular Biology 2016
Bioinformatics 5: (2 points/question)
12. Submit the annotated sequence (nucleotides with corresponding amino acids) of the longest
ORF found for the viral1 sequence.
1715 atgaaagtaaaactactgatcctgttatgtacatttacagctaca
M K V K L L I L L C T F T A T
1670 tatgcagacacaatatgtataggctaccatgccaacaactcaacc
Y A D T I C I G Y H A N N S T
1625 gacactgttgacacagtacttgagaagaatgtgacagtgacacac
D T V D T V L E K N V T V T H
1580 tctgtcaacctacttgaggacagtcacaatggaaaactgtgccta
S V N L L E D S H N G K L C L
1535 ctaaaaggaatagcccccctacaattgggtaattgcagcgttgcc
L K G I A P L Q L G N C S V A
1490 ggatggatcttaggaaacccagaatgcgaattactgatttccaag
G W I L G N P E C E L L I S K
1445 gaatcatggtcctacattgtagaaacaccaaatcctgagaatgga
E S W S Y I V E T P N P E N G
1400 gcatgttacccagggtatttcgccgactatgaggagctaagggag
A C Y P G Y F A D Y E E L R E
1355 caattgagttcaatatcttcatttgagagattcgaaatattcccc
Q L S S I S S F E R F E I F P
1310 aaagaaagctcatggcccaaccacaccgtaaccggagtatcagca
K E S S W P N H T V T G V S A
1265 tcatgctcccataatgggaaaagcagtttttacagaaatttgcta
S C S H N G K S S F Y R N L L
1220 tggctgacggggaagaatggtttgtacccaaacctgagcaagtcc
W L T G K N G L Y P N L S K S
1175 tatgcaaacaacaaagagaaagaagtccttatactatggggtgtt
Y A N N K E K E V L I L W G V
1130 catcacccgcctaacataggggaccaaaggaccctctatcacaca
H H P P N I G D Q R T L Y H T
1085 gaaaatgcttatgtctctgtagtgtcttcacattatagcagaaga
E N A Y V S V V S S H Y S R R
1040 ttcaccccagaaataaccaaaaggcccaaagtaagagatcaggaa
F T P E I T K R P K V R D Q E
995 ggaagaatcaactactactggactctgctggaacccggggataca
G R I N Y Y W T L L E P G D T
950 ataatatttgaggcaaatggaaatctaatagcgccatggtatgct
I I F E A N G N L I A P W Y A
905 ttcgcactgagtagaggctttggatcaggaatcatcacctcaaat
F A L S R G F G S G I I T S N
860 gcaccaatggatgaatgtgatgctaagtgtcaaacacctcaggga
A P M D E C D A K C Q T P Q G
815 gctataaacagcagtcttcctttccagaatgtacacccagtcaca
A I N S S L P F Q N V H P V T
770 ataggagagtgtccaaagtatgtcaggagtgcaaaattaaggatg
I G E C P K Y V R S A K L R M
725 gttacaggactaaggaacatcccatccattcaatccagaggtttg
V T G L R N I P S I Q S R G L
680 tttggagccattgccggtttcattgaaggggggtggactggaatg
F G A I A G F I E G G W T G M
635 gtagatgggtggtatggttatcatcatcagaatgagcaaggatct
V D G W Y G Y H H Q N E Q G S
590 ggctatgctgcagatcaaaaaagcacacaaaatgccattaacggg
G
Y
A
A
D
Q
K
S
T
Q
N
A
I
N
G
13. Does the sequence submitted in the previous question represent the genome sequence of the
virus, the mRNA sequence or both? mRNA
Molecular Biology 2016
14. Does the original sequence on which the analysis was performed represent the genome
sequence of the virus, the mRNA sequence or both? Genome
15. Submit the following information for the longest ORF from the viral1 and viral2 sequences:




The definition
Influenza A virus (A/New York/492/2003(H1N2)) segment 4, complete sequence &
Influenza A virus (A/New York/492/2003(H1N2)) segment 6, complete sequence.
The organism this gene comes from
Influenza A virus (A/New York/492/2003(H1N2)) for both
The name of the gene (Not the gene product)
HA & NA
The name of the gene product
hemagglutinin & neuraminidase
16. Submit the following information for the viral3 sequences:




What SNPs (single nucleotide polymorphisms) has the viral3 sequence acquired?
Indel –C at position 1537 and + A at position 1600
What is the percentage identity at the nucleotide level between the viral1 and viral3
sequences? 100%
What is the percentage identity at the protein level between the two sequences? 98.83%
Indicate the number of conserved (2), semi-conserved (1) and non-conserved amino acid
(1) changes.
17. What three conserved protein domains are present in the unknown human sequence?
PTPc, Ig3_RPTP_IIa_LAR_like, and Ig2_RPTP_IIa_LAR_like
Molecular Biology 2016
Assignment #4
(3 points/question)
1. Indicate how each of the following conditions would affect stringency: increase, decrease or
no effect.
Condition
Effect
Increased temperature
Increase
Increased NaCl concentration
Decrease
Higher G:C content
No effect
Higher urea concentration
Increase
Higher probe concentration
No effect
2. Below are the sequences of portions of the yfg1 mRNA and three non-yfg1 mRNAs found in
zebrafish embryos (A, B, and C).
5’...GAUGAAAGAUCAGGUCUGAAUGUAU...3’ yfg1 mRNA
5’...UUUGAAAGAUCAGGUCUGAAUGUAU...3’ A
5’...CUACUUUCUAGUGGUCUGAAUGUAU...3’ B
5’...CUACUUUCUAGUCCUCUGAAUGUAU...3’ C
You’ve created a probe which is 100% complementary to the sequence of the yfg1 mRNA:
5’...TTCAGACCTGATCTTT...3’. You hybridize the probe to a blot of total zebra fish RNA
at room temperature (20˚C), and then want to adjust the stringency of your washes by
increasing the temperature of the buffer. You perform your highest stringency wash at a
temperature of 50˚C. Which of the four mRNA(s) will you “see” as bands on film when you
develop your Northern blot?
A.
B.
C.
D.
E.
F.
yfg1 alone
yfg1 and A
yfg1 and B
yfg1 and C
all of the above
none of the above
3. Starting with a single molecule of an mRNA template and sufficient primers, enzyme and all
other co-factors for successful gene specific RT-PCR, how may PCR cycles are required to
have 8 molecules of products with ends defined by both primers? By this I mean that you
count only double stranded molecules that begin and end at the primer binding sites but lack
any other sequence. 5 cycles
Molecular Biology 2016
4. One strand of the extremely tiny gene Liliputian is shown below, with its start and stop
codons underlined.
5’-TGAGGCATCATCGGTATGGCACCCTTAATGGGCATTGCACCCATAGTACGATAAGCATGTCCTGAA-3’
Is this the template or the non-template strand? non-template
5. I want to use RT-PCR to make a copy of the entire Liliputian gene. Indicate, the sequence of
a primer of 6 nucleotides that could be used for the first strand synthesis of cDNA. 5’TTCAGG
Refer to the following description to answer questions 6-10
Below are 210 consecutive base pairs of DNA that includes only the beginning of the sequence of
gene X. The underlined sequence (from position 20-54) represents the promoter for gene X and
the underlined and italicized sequence (from position 71-90) encodes the gene X ribosome binding
(RBS) site. Transcription begins at and includes the T/A base pair at position 60 (underlined).
1
10
20
30
40
50
60
70
I--------I---------I---------I---------I---------I---------I---------I
5’ ATCGGTCTCGGCTACTACATAAACGCGCGCATATATCGATATCTAGCTAGCTATCGGTCTAGGCTACTAC
3’ TAGCCAGAGCCGATGATGTATTTGCGCGCGTATATAGCTATAGATCGATCGATAGCCAGATCCGATGATG
Promoter
80
90
100
110
120
130
140
I---------I---------I---------I---------I---------I---------I
5’ CAGGTATCGGTCTGATCTAGCTAGATGCTCTTCTCTCTCTCCCCCGCGGGGGCTGTACTATCATGCGTCG
3’ GTCCATAGCCAGACTAGATCGATCTACGAGAAGAGAGAGAGGGGGCGCCCCCGACATGATAGTACGCAGC
RBS
150
160
170
180
190
200
210
---------I---------I---------I---------I---------I---------I---------I
5’ TCTCGGCTACTACGTAAACGCGCGCATATATCGATATCTAGCTAGCTATCGGTCTCGGCTACTACGTAAA
3’ AGAGCCGATGATGCATTTGCGCGCGTATATAGCTATAGATCGATCGATAGCCAGAGCCGATGATGCATTT
6. What are the first 6 nucleotides of the mRNA from gene X? TAGGCT
7. What are the first 4 amino acids encoded by gene X? Met-Leu-Phe-Ser
8. You have found a mutant of gene X. The mutation represents an SNP which changes the T/A
base pair at position 110 (underlined) to A/T. Would the mRNA expressed from this version
of gene X be longer, shorter, or the same as that produced from the normal gene X? Same
9. If the mRNA can be translated, would you expect the protein to be longer, shorter, or the same
as that produced from the normal gene X? Same
10. Do you expect that the protein produced will have a similar activity as compared to the normal
protein X? Yes
Molecular Biology 2016
11. You have been asked to PCR amplify a specific sequence from cDNA that was synthesized
from mRNA isolated from brain tissue. After you run your potential PCR product on an agarose
gel containing ethidium bromide, you observe no bands when you visualize the gel using
ultraviolet light. Why might this be the case?
A. The gene you are interested in is not expressed in brain tissue.
B. An oligo dT was used instead of a oligo dA to prime the first strand cDNA synthesis.
C. A specific primer whose sequence was that of the non-coding strand of the gene was used
to prime the first strand cDNA synthesis.
D. You used reverse transcriptase instead of Taq polymerase to synthesize the first strand of
cDNA.
Choose all possible answers.
12. The following schematic represents a yeast gene and it's various elements:
(200)
(1)
ATG (510)
(1060)
(1600)
(1850)
(2110)
TAG (2510)
(2890)
Promoter
Transcription terminator
Exon
Indicate the predicted sizes of each of the following:
A.
B.
C.
D.
Pre-mRNA (non processed) 2690b
mRNA 1890b
Protein 399 aa
mRNA from a mutant gene with a point mutation at position 1698 creating a stop codon
1890b
E. Protein from a mutant gene with a point mutation creating a stop codon at position 1700
215 aa
Molecular Biology 2016
13. The gene described in the previous question is known to be repressed 5 fold when cells are
grown in glucose as compared to growth in glycerol. To study the expression of this gene, a
northern analysis was performed for a wild type strain as well as various mutants grown in
glycerol. Blots were simultaneously probed for the gene shown above, as well as a house
keeping gene; GAPDH. A densitometric analysis was then performed. Some of the results
obtained are presented in the table below:
Densitometric
data
Strain and growth condition
Yeast GAPDH
gene
500
500
1 Wild type grown in glucose
100
2 Wild type grown in glycerol
500
250
3 Mutant strain with a 1 base insertion at position 515 grown in glycerol
1250
4 Mutant strain with an snp at position 515 creating a neutral amino acid
250
1250
change grown in glycerol
5 Mutant strain with an snp at position 515 creating non-synonymous 500
2500
non conserved amino acid change grown in glycerol
6 Mutant strain with a mutation which enhances the promoter's activity
500
5000
2 fold grown in glycerol
Complete the table to indicate the approximate densitometric values expected for the yeast gene's
mRNA.
14. Which of the above strains grown in glycerol would you expect to have the highest level of
specific activity (activity per protein molecule)? 2, 4, and 6
15. Which of the above strains grown in glycerol would you expect to have the lowest level of
specific activity (activity per protein molecule)? 3
16. Which of the above strains under which growth condition would you expect to synthesize the
highest level of protein per cell (whether active or inactive) 6
Molecular Biology 2016
Lab Exercises
(4 points/question)
RT-PCR
1. Submit a figure of your RT-PCR gel with an appropriate figure legend. Your figure legend
must include the sizes of the products observed in each lane. See general directives
2. What was the purpose of the DNAse treatment? Remove contaminating genomic DNA
3. What was the purpose of the PCR reaction of the PCR reaction of RNA NOT treated with
either DNAse or RT? Assess whether products are from RNA or DNA
4. Did the the sizes of RT specific PCR products correspond to that of the PCR using genomic
DNA as template? If it did, indicate what that tells you about this RNA. If it didn’t, indicate
what that tells you about this RNA. If yes no introns, if no, gene has an intron
Project IV: Control of GFP (northern analysis/enzyme activity)
5. Submit a figure of your RNA gel and corresponding northern hybridization with an appropriate
figure legend. See general directives
6. Obtain densitometric data for the GFP and bla mRNA signals obtained from the E.coli
recombinant cultures grown under the different growth conditions. Submit a table of the raw
data obtained with Image J. (See tutorial on this course's web site under the heading "Bioinfo
links") Your table must include the raw data (the values for each of the areas), the normalized
values (GFP reading/bla reading) for each of the growth conditions and the relative expression
as compared to growth of the wild type GFP recombinant grown in glucose. See general
directives
7. Submit a table of the data and corresponding analysis of the GFP activity assays. Your table
should include: See general directives

The growth condition and the type of mutation of each strain assayed.

Absorbencies and OD600 values for each of the strains under each of the growth conditions
assayed.

Normalized activity units (GFP absorbance/1 OD600 value) for each of the strains under
each of the growth conditions assayed.

The relative activity levels as compared to growth of the wild type GFP recombinant grown
in glucose.

A caption indicating which mutant showed the greatest relative reduction in activity.
Molecular Biology 2016
Bioinformatics: (2 points/question)
You should now be quite familiar with the NCBI site and be able to complete the following
exercise with relatively few directives. Consider this as a practice run for the bioinformatics
section on the final exam.
CAGGCTCCAGAACACCACCATTGGGTTAACTGTGTTTGCCATCAAGAAATACGTGGCTTTCCTGAAGCT
GTTCCTGGAGACGGCGGAGAAGCACTTCATGGTGGGCCACCGTGTCCACTACTATGTCTTCACCGACCAG
CCGGCCGCGGTGCCCCGCGTGACGCTGGGGACCGGTCGGCAGCTGTCAGTGCTGGAGGTGCGCGCCTACA
AGCGCTGGCAGGACGTGTCCATGCGCCGCATGGAGATGATCAGTGACTTCTGCGAGCGGCGCTTCCTCAG
CGAGGTGGATTACCTGGTGTGCGTGGACGTGGACATGGAGTTCCGCGACCACGTGGGCGTGGAGATCCTG
ACTCCGCTGTTCGGCACCCTGCACCCCGGCTTCTACGGAAGCAGCCGGGAGGCCTTCACCTACGAGCGCC
GGCCCCAGTCCCAGGCCTACATCCCCAAGGACGAGGGCGATTTCTACTACCTGGGGGGGTTCTTCGGGGG
GTCGGTGCAAGAGGTGCAGCGGCTCACCAGGGCCTGCCACCAGGCCATGATGGTCGACCAGGCCAACGGC
ATCGAGGCCGTGTGGCACGACGAGAGCCACCTGAACAAGTACCTGCTGCGCCACAAACCCACCA
8. What is the name of the gene from which this sequence was obtained? ABO
9. What is the probability that the gene found in question 1 is a true match? 0
10. Is the source sequence RNA or genomic DNA? Genomic DNA
11. What is the accession number of the gene found in question 1? LN680546
12. What is the accession number of the protein corresponding to the gene found in question 1?
CEJ95649
13. Give the accession number of the chimpanzee (Pan troglodytes) protein orthologue of the
protein found in question 3. NP_001181854
14. What is the percent identity between the protein from question 3 and the rat protein orthologue
obtained for question 4? 98.02%
15. How many times do BamHI and HincII cut within the gene obtained in question 1? 0 and twice
respectively
16. Which of the following primers would hybridize to the mRNA of the gene obtained in question
1 which would allow you to perform a reverse transcriptase reaction?
A.
B.
C.
D.
CCGCAACACCTCGGC
CAAGAACCACCAGGC
CCGCAACACCTCGAA
GTGCCCAGCAGCTGC
Molecular Biology 2016
15. The sequence at the bottom of this page represents a different allele of the gene discussed in
question 1. Does it contain one or more SNPs as compared to the sequence corresponding to
that obtained from the NCBI site? If so, what are they? Indicate the position as well as the base
change (Ex. G218 to C) Indel of G at pos. 53, SNP G298 to A
16. Do these SNPs change the reading frame of the gene? Yes
17. How long is the protein encoded by the gene sequence obtained in question 1? 354 aa
GGAGGCCGAGACCAGACGCGGAGCCATGGCCGAGGTGTTGCGGACGCTGGCCGGGAAAACCAAAATGCCA
CGCACTTCGACCTATGATCCTTTTCCTAATAATGCTTGTCTTGGTCTTGTTTGGTTACGGGGTCCTAAGC
CCCAGAAGTCTAATGCCAGGAAGCCTGGAACGGGGGTTCTGCATGGCTGTTAGGGAACCTGACCATCTGC
AGCGCGTCTCGTTGCCAAGGATGGTCTACCCCCAGCCAAAGGTGCTGACACCGTGTAGGAAGGATGTCCT
CGTGGTGACCCCTTGGCTAGCTCCCATTGTCTGGGAGGGCACATTCAACATCGACATCCTCAACGAGCAG
TTCAGGCTCCAGAACACCACCATTGGGTTAACTGTGTTTGCCATCAAGAAATACGTGGCTTTCCTGAAGC
TGTTCCTGGAGACGGCGGAGAAGCACTTCATGGTGGGCCACCGTGTCCACTACTATGTCTTCACCGACCA
GCCGGCCGCGGTGCCCCGCGTGACGCTGGGGACCGGTCGGCAGCTGTCAGTGCTGGAGGTGCGCGCCTAC
AAGCGCTGGCAGGACGTGTCCATGCGCCGCATGGAGATGATCAGTGACTTCTGCGAGCGGCGCTTCCTCA
GCGAGGTGGATTACCTGGTGTGCGTGGACGTGGACATGGAGTTCCGCGACCACGTGGGCGTGGAGATCCT
GACTCCGCTGTTCGGCACCCTGCACCCCGGCTTCTACGGAAGCAGCCGGGAGGCCTTCACCTACGAGCGC
CGGCCCCAGTCCCAGGCCTACATCCCCAAGGACGAGGCCGATTTCTACTACCTGGGGGGGTTCTTCGGGG
GGTCGGTGCAAGAGGTGCAGCGGCTCACCAGGGCCTGCCACCAGGCCATGATGGTCGACCAGGCCAACGG
CATCGAGGCCGTGTGGCACGACGAGAGCCACCTGAACAAGTACCTGCTGCGCCACAAACCCACCAAGGTG
CTCTCCCCCGAGTACTTGTGGGACCAGCAGCTGCTGGGCTGGCCCGCCGTCCTGAGGAAGCTGAGGTTCA
CTGCGGTGCCCAAGAACCACCAGGCGGTCCGGAACCCGTGAGCGGCTGCCAGGGGCTCTGGGAGGGCTGC
CGGCAGCCCCGTCCCCCTCCCGCCCTTGGTTTTAGCAGAACGGGTAAACTCTGTTTCCTTTGTCCGTCCT
GTTGTGAGTAACTGAAGCCTAGGCCCCGTCCCCACCTCAAATCACACACACCCCCTCCCCACCACAGAGA
CACCATTACATACACAGACACACACAGAAAGACACACACAGACACAAAATCACACACACACCCTCCCCGC
CACAGAGACACCATTACATACACAGACACACACAGAAAGACACAGACACAAAATCACACACACACCCTCC
CCGCCACAGAGACACACCATTACATACACAGACACGCAATCGCAGATACGCCCTTCCGGCCACAGAAACA
CACCATTACACACACATACACAGAAAGACACACACAGACACACAATCACACGCAGCCCCTCCCCGCCACA
GAGACACACCATTACATACACAGACACACACAGAAAGACAC